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Biomedical subjects

J A Shafer

Publications and source records attributed to J A Shafer.

At least 91 records · Page 5Linked to original sources
PubMed1984

A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit.

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.

Blood Coagulation Tests↗
PubMed1984

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin.

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.

Cell Membrane↗
PubMed1984

Fluorescence energy transfer studies on lima bean lectin. Distance between the subunit hydrophobic binding site and the thiol group essential for carbohydrate binding.

Measurements of the efficiency of singlet-singlet energy transfer were used to determine the distance between the hydrophobic binding site and the thiol group required for carbohydrate-binding activity of lima bean lectin. 1-Anilino-8-naphthalenesulfonate, bound to the hydrophobic binding site by noncovalent interactions, was used as the donor. Two different nonfluorescent probes were used as the acceptors: a mercurial, 2-chloromercuri-4-nitrophenol, and a maleimide, 4-dimethylaminophenylazophenyl-4'-maleimide. Acceptor was covalently attached to the thiol group at the putative carbohydrate binding site. The efficiency of energy transfer in both the 1-anilino-8-naphthalenesulfonate/2-chloromercuri-4-nitrophenol and and 1-anilino-8-naphthalenesulfonate/4-dimethylaminophenylazophenyl-4' -maleimide donor-acceptor systems indicated an apparent distance of 28 A between the two sites, assuming that the transition dipole of the donor is not correlated with respect to that of the acceptor and that each donor is quenched by a single acceptor. Using an alternate model wherein each donor is equally quenched by two acceptors on adjacent subunits, an apparent distance of 33.4 A was calculated. The fact that two donor-acceptor pairs with different Förster's critical distance parameters yielded the same distance between the sites is consistent with our assumption of uncorrelated donor-acceptor transition dipoles.

Binding Sites↗
PubMed1983

Promotion of thrombin-catalyzed activation of factor XIII by fibrinogen.

High-performance liquid chromatography was used to analyze the kinetics of the thrombin-catalyzed release of the activation peptide from the factor XIII zymogen (fibrin-stabilizing factor). The specificity constant (kcat/Km) for this reaction, measured at factor XIII concentrations much below Km, was (0.13-0.16) X 10(6) M-1 s-1 at pH 7.4, mu = 0.15, and 37 degrees C. Separate estimates, obtained from the dependence of the initial rates of release of the activation peptide on the concentration of factor XIII, gave values of 10 (+/- 3) s-1 for kcat and 84 (+/- 30) microM for Km, in terms of ab protomers of the zymogen. The thrombin-mediated release of the activation peptide was dramatically enhanced in the presence of fibrinogen. Furthermore, the time course of release, in relation to that of fibrinopeptide A, suggested that some des-A-fibrinogen species (e.g., alpha 2B beta 2 gamma 2) may be the true activator for promoting the cleavage of the Arg-36 peptide bonds in the a subunits of factor XIII. This observation suggests that generation of factor XIIIa and its substrate (fibrin) is coordinated so that thrombin-mediated zymogen activation proceeds efficiently only after the process of clotting has been initiated by the removal of fibrinopeptide A from fibrinogen.

Blood Coagulation↗
PubMed1983

Steady state kinetic parameters for the thrombin-catalyzed conversion of human fibrinogen to fibrin.

Steady state kinetic parameters were evaluated for the hydrolytic release of fibrinopeptides A and B (FPA and FPB) from human fibrinogen by human thrombin at pH 7.4, 37 degrees C, and gamma/2 0.15. At low concentrations of fibrinogen (less than 0.4 microM), the release of FPA from A alpha-chains was first order with respect to both the concentration of fibrinogen A alpha-chains and thrombin. The second order rate constant yielded a value of 11.6 (+/- 0.3) X 10(6) M-1 S-1 for the specificity constant (kcat/Km) for this process. Values of 84 (+/- 4) S-1 and 7.2 (+/- 0.9) microM were evaluated for kcat and Km for the thrombin-catalyzed release of FPA from normal human fibrinogen. The amino acid replacement ArgA alpha 16 leads to His present in fibrinogen Petoskey was shown to result in a 160-fold decrease in the specificity constant for hydrolysis at A alpha 16 and concomitant release of FPA. A kinetic analysis for determination of the sequentiality of release of fibrinopeptides was presented. It indicated that at least 97% of FPB was released after FPA. The specificity constant for release of FPB from intact fibrinogen (if it occurs) was less than 3% of that for release of FPA and less than 10% of that for release of FPB from des-A fibrinogen. The specificity constant for the release of FPB from des-A fibrinogen was 4.2 (+/- 0.2) X 10(6) M-1 S-1. The polymerization inhibitors EDTA and Gly-Pro-Arg-Pro inhibited release of FPB but not FPA. These observations are consistent with the generally accepted view that the predominant pathway for the conversion of normal human fibrinogen to fibrin is one wherein FPA is released, des-A fibrinogen polymerizes, and then FPB is released.

Edetic Acid↗
PubMed1983

Characterization of the catalytic pathway for D-serine dehydratase. Evidence for variation of the rate-determining step with substrate structure.

Steady state kinetic parameters (kcat and Km) which were determined for the D-serine dehydratase-catalyzed decomposition of the isomers of serine and threonine at pH 5.7, 7.8, and 8.9 indicated that the enzyme exhibited considerable kinetic specificity. At pH 7.8 and 25 degrees C, the specificity constants (kcat/Km) were as follows: D-serine (3.4 X 10(5) M-1 s-1), D-threonine (2.9 X 10(4) M-1 s-1), D-allothreonine (7.5 X 10(3) M-1 s-1), and L-serine (38 M-1 s-1). Substrate C-2 deuterium atom isotope effects on the steady state kinetic parameters disclosed that the rate-determining step in the catalytic pathway varied with substrate structure and pH. Removal of the C-2 hydrogen atom of the substrate was shown to be fully rate-determining with L-serine and partially rate-determining with D-allothreonine as substrate at pH 7.8. Stopped flow measurements of absorbance and fluorescence were used to characterize intermediates in the catalytic pathway. These measurements indicated that D-serine, in addition to being the best substrate, was processed faster than the other substrates through steps in the catalytic pathway which were not rate-controlling. Kinetic evidence also was obtained which indicated that the base which accepts the proton from the C-2 carbon atom of the substrate must be aprotic. Thus, the catalytic site of D-serine dehydratase should contain a basic group in addition to the active site lysyl residue.

Escherichia coli↗
PubMed1983

A reaction pathway for transimination of the pyridoxal 5'-phosphate in D-serine dehydratase by amino acids.

Transimination of the enzyme-linked cofactor by an amino acid is the first chemical transformation in the reactions catalyzed by pyridoxal 5'-phosphate-requiring enzymes. In this work, stopped flow fluorimetry was used to characterize the kinetics of transimination of the cofactor in D-serine dehydratase by several amino acids. The results of these studies indicate that transimination is a multistep process, the first step of which is probably formation of a noncovalent complex between the enzyme and the amino acid. D-Serine dehydratase was found to exhibit considerable specificity in the transimination reaction. Furthermore, the enzyme was shown to facilitate the transimination reaction with amino acids and inhibit transimination of the bound cofactor by amines lacking a carboxylate group. A reaction pathway was proposed for the transimination process which accounts for the specificity of the enzyme and indicates the changes in the conformation of the bound cofactor as dictated by the stereoelectronic requirements of the transimination reaction.

Amino Acids↗
PubMed1983

Stoichiometry for the binding of insulin to insulin receptors in adipocyte membranes.

Insulin receptor molecules in rat adipocyte plasma membranes were shown to be monovalent with respect to their capacity to bind insulin. The 1:1 stoichiometry for insulin binding was determined by a "double-probe labeling" procedure, wherein 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess saturating concentration of an unlabeled biotinylated insulin derivative (probe 2). If the receptor were competent to bind more than one insulin molecule, any receptor molecule that was cross-linked to probe 1 also should have been cross-linked to probe 2 in the double probe labeling procedure. The monovalent character of the insulin receptor was indicated by the failure of the probe 1-linked receptor to be cross-linked to probe 2. This was indicated by the failure of succinylavidin to increase the molecular weight of the probe 1-linked receptor. Control experiments indicated that succinylavidin increased the molecular weight of receptor that had been cross-linked to probe 2. The 1:1 stoichiometry for insulin binding demonstrated here indicates that if insulin receptors contain more than one insulin binding subunit, the binding of insulin to its receptor must be a highly negatively cooperative process.

Adipose Tissue↗
PubMed1982

A kinetic method for characterization of heterogenous fibrinogen and its application to fibrinogen Grand Rapids, a congenital dysfibrinogenemia.

A kinetic analysis was developed to determine the steady state kinetic parameter Kcat/KM for the thrombin-catalyzed release of FPA from abnormal and normal fibrinogen in mixtures of the two. Such mixtures are likely to comprise the fibrinogen of individuals with congenital dysfibrinogenemia. The analysis was used to characterize fibrinogen Grand Rapids a new congenital dysfibrinogenemia. It indicated that fibrinogen from affected individuals was composed of normal and abnormal fibrinogen in roughly equal amounts, and that the value of kcat/KM for the thrombin-catalyzed release of FPA from the fibrinogen variant was 77-fold lower than that for the release of FPA from the normal fibrinogen. In separate studies, fibrinogen Grand Rapids was found to exhibit a reduced clottability. Additionally, affected individuals appeared to have plasma fibrinogen concentrations which were about one-third the normal value.

Blood Coagulation Disorders↗
PubMed1981

Inactivation of D-serine dehydratase by alkylamines via a transimination of enzyme-linked cofactor.

The time-dependent inactivation of D-serine dehydratase by alkylamines was characterized. Evidence is presented indicating that inactivation proceeds via a transimination reaction analogous to the first step in the catalytic pathway. The product of alkylamine attack, an alkylamine pyridoxal 5'-phosphate Schiff base, readily dissociated from D-serine dehydratase to produce inactive apoenzyme. Reaction with alkylamines was shown to be a convenient way of producing apoenzyme which can be reconstituted with pyridoxal 5'-phosphate to fully active holoenzyme. Amino acids such as glycine and alanine, unlike alkylamines, did not resolve D-serine dehydratase but were competitive inhibitors of the enzyme. The inability of amino acids to resolve D-serine dehydratase from its cofactor was attributed to a failure of the amino acid-cofactor Schiff base to dissociate from the enzyme. Transimination of D-serine dehydratase with its substrate D-serine was at least 3.5 X 10(5) times faster than a nonenzymic model transimination reaction and more than 70 million times faster than the reaction of the enzyme with 2-hydroxyethylamine, indicating that the carboxyl group of the substrate is an important structural determinant for catalysis of the transimination step in the catalytic pathway. An analysis of the inhibitory effect of potassium ion on the rate and extent of inactivation of D-serine dehydratase by alkylamines indicated that K+ binding increased the affinity of the enzyme for its cofactor by at least 13-fold.

Amines↗
PubMed1981

Determination of a low pK for histidine-159 in the S-methylthio derivative of papain by proton nuclear magnetic resonance spectroscopy.

Proton NMR spectroscopy was used to study the ionization behavior of His-159 in a derivative of papain (papain-S-SCH3). In this catalytically inactive derivative of papain, the active-site thiol group of Cys-25 is S-methyl-thiolated so that it cannot form a thiolate anion. The pH dependence of the chemical shift of the C epsilon 1 H resonance of His-159 indicated a pK of 3.45 +/- 0.07 at 45 degrees C in 2H2O with no added ions other than those required for titration. In acetate buffers at an ionic strength of 0.05, the pK increased to 3.87 +/- 0.12. Conversion of papain-S-SCH3 to active papain at pH* 4.17 (at 45 degrees C and an ionic strength of 0.05) caused the position of the C epsilon 1 H resonance to change from a position indicative of partial protonation of His-159 to a position indicative of full protonation, consistent with the existence of an imidazolium-thiolate ion-pair interaction between His-159 and Cys-25 in the active enzyme.

Histidine↗
PubMed1981

Effect of cysteine-25 on the ionization of histidine-159 in papain as determined by proton nuclear magnetic resonance spectroscopy. Evidence for a his-159--Cys-25 ion pair and its possible role in catalysis.

Papain was succinylated in order to increase its solubility above pH 8 so that proton NMR spectroscopy could be used to study the ionization of His-159 at the active site of the enzyme. The pH dependence of NMR spectra of catalytically active succinyl-papain and the methylthio derivative of the active-site cysteinyl residue of succinyl-papain (succinyl-papain-S-SCH3) were determined between pH 6 and 10. The pH dependence of the C epsilon 1 H resonance of His-159 in catalytically active succinyl-papain indicates that His-159 has a pK of about 8.6 in the catalytically active form of the enzyme. The position of this resonance in succinyl-papain-S-SCH3 indicates that when the active-site cysteinyl residue is methylthiolated, His-159 is completely deprotonated between pH 6 and 10. This result is taken as evidence for an imidazolium--thiolate ion-pair interaction between His-159 and Cys-25 wherein neutralization of the charge on the thiolate anion by methylthiolation would be expected to cause a marked decrease in the pK of His-159. A possible catalytic role for the ion pair in the acylation step in papain-catalyzed reactions is proposed wherein attack of a substrate by the imidazolium--thiolate ion pair is accompanied by an increase in the acidity of the imidazolium group that facilitates expulsion of the leaving group of the substrate.

Binding Sites↗
PubMed1981

Perturbations in the free energy and enthalpy of ionization of histidine-159 at the active site of papain as determined by fluorescence spectroscopy.

Fluorometric titrations of papain, succinyl-papain, and the corresponding methylthio derivatives of Cys-25 (papain-S-SCH3 and succinyl-papain-S-SCH3) were determined. Removal of the methylthio group from Cys-25 resulted in an increase of approximately 4 pK units in the fluorometrically determined pK value. The correspondence between the ionization behavior as determined by proton NMR and fluorometric titrations indicated that fluorescence titrations reflect the ionization behavior of His-159 in both the active enzyme and the methylthio derivative. The ionic strength dependence of the pK was analyzed in terms of simple electrostatic theory and was shown to be consistent with the charge on the protein. The temperature dependence of the pK values of His-159 indicated an increase in the heat of ionization from about 0 to 8 kcal/mol upon removal of the methylthio blocking group from Cys-25. Measurements of the effect of solvent on the pK's and heats of ionization of simple model compounds indicated that the observed shift in enthalpy of ionization of His-159 upon removal of th methylthio group from Cys-25 is not unreasonable in light of the accompanying perturbation is more than 4 pK units in the pK of His-159. The perturbations in enthalpies and free energies are attributed to formation of an ion pair. The ionization behavior of His-159 in thiol-blocked derivatives of papain is consistent with the involvement of His-159 in the deacylation step in papain catalysis.

Benzoates↗